The physiological turnover of articular cartilage represents a fine balance between synthesis and degradation. It is a feature of normal growth and development and maintenance of cartilage in the adult. Net cartilage destruction with ensuing loss of joint function is a feature of the arthritides. An understanding of the factors mediating cartilage breakdown and its control, and the ability to detect and measure destruction, is therefore of great importance, since from this an approach can be made to monitor disease activity at the level of cartilage breakdown, and design therapies for the reduction of pathological cartilage destruction and the enhancement of repair in this tissue. Progress to this goal is dependent on identifying the degradative events occurring in articular cartilage and correlating these with the many degradative agents potentially active in the tissue.
The destruction of articular cartilage is due, in part, to the degradation of the extracellular matrix. Type II collagen constitutes the bulk of the fibrillar backbone of cartilage matrix, just as type I collagen forms the fibrillar organization of the extracellular matrix of most other tissues such as skin, bone, ligaments and tendons. These collagens are composed of a tightly wound triple helix. In articular cartilage, type II collagen fibrils are responsible for the tensile strength whereas the proteoglycans provide the compressive stiffness necessary for normal articulation and function. The precise mechanisms by which these connective tissue components are degraded are not fully understood (11). In mammals, one mechanism involves collagenases, enzymes capable of a site-specific cleavage of helical collagen.
Incapable of maintaining a helical structure at physiological temperatures, collagenase-cleaved fibrillar collagens unwind and become susceptible to further degradation by other proteinases in the extracellular space. In this regard, collagenases can be considered the rate limiting enzymes involved in collagen degradation. There are three types of human collagenase that are known to cleave the human fibrillar type I, and II collagens. They are the matrix metalloproteinases (MMP): MMP-1 (interstitial collagenase or collagenase-1); MMP-8 (neutrophil collagenase or collagenase-2) and MMP-13 (collagenase III). Collagenases cleave type II collagen between residues 775 and 776 to produce the characteristic 1/4 and 3/4 length .alpha.-chain fragments that are identifiable by polyacrylamide gel electrophoresis. Once cleaved by collagenase, the three strands of the collagen molecule begin to unwind and become susceptible to further degradation by the same and other proteinases in the extracellular space. Cleavage results in the release of fragments of type II collagen which can be detected in culture media. In vivo, these fragments are diffused into body fluids. They are present in the synovial fluid from which they may enter the lymphatics and drain eventually into peripheral blood. Fragments may also enter urine following filtration in the kidney.
Detection and measurement of arthritis-related type II collagen derived degradation products poses difficulty for clinicians. Traditional methods for the detection of collagen loss from cartilage have relied on the use of stains such as van Gieson's. The latter was used by Fell and her collaborators (2) to detect collagen loss in cartilage which results in a loss of the normal bright-pink staining. This method, like others of its kind, is, however, of unproven specificity. At the present time, damage to cartilage in joints is recorded by X-ray which reveals a loss of joint space as cartilage is destroyed and lost. This change is a particular feature of osteoarthritis and of rheumatoid arthritis. X-ray does not constitute a particularly sensitive tool for diagnosis of arthritis, as a significant amount of cartilage damage must occur before it becomes detectable by this method. As such, X-ray cannot be used to measure early damage to cartilage. Furthermore, X-ray cannot usually be used in studies of less than one year (rheumatoid arthritis) or two years (osteoarthritis) in duration to monitor the subtle effects of drug treatment on cartilage breakdown. This is a particularly significant problem as recent research indicates that drug treatment of arthritis symptoms (principally pain and inflammation) may actually exacerbate cartilage degradation over time. As such, it has become more important to monitor the direct effects of the disease and of drug treatment on cartilage. This is especially necessary since loss of joint function in arthritis results primarily from skeletal damage, particularity to cartilage.
Attempts have been made to develop methods to detect cartilage breakdown, other than by X-ray. However, initially immunogenic techniques were not promising as, for a long time, collagenous proteins were considered non-immunogenic. Furthermore, monoclonal antibodies against collagens often are only capable of binding a low percentage of antigen, i.e. they demonstrate partial binding (52). Dodge et al (1989) (1) described a polyclonal antibody to an epitope on unwound type II collagen. However, polyclonal antibodies prepared in animals have the disadvantage of having a heterogenous composition and may have a different reactivity from animal to animal.
U.S. Pat. No. 5,140,103 (Eyre, Aug. 18, 1992) and U.S. Pat. No. 5,641,837 (Eyre, Jun. 24, 1997) describe a method of determining collagen degradation by detection of a C-terminal type II collagen telopeptide containing a hydroxylysyl pyridinoline cross-link. However, the telopeptide antigen detected by this assay is not produced by cleavage with collagenase, the enzyme believed to be directly responsible for arthritis-related collagen breakdown as shown recently by Billinghurst et al (49).
Accordingly, a need exists for an assay to detect and measure degradation of type II collagen caused by collagenase so as to provide a mechanism for early detection of arthritis and joint damage and for monitoring disease activity, progression and the efficacy of arthritis treatment. In particular, there is a need for such an assay which can be performed using body fluids such as urine, synovial fluid and serum.